Pages

Monday, 19 November 2012

Stanford University School of Medicine scientists have demonstrated, in a study conducted jointly with researchers at Yale University, that induced-pluripotent stem cells — the embryonic-stem-cell lookalikes whose discovery a few years ago won this year's Nobel Prize in medicine — are not as genetically unstable as was thought.

The new study, which will be published online Nov. 18 in Nature, showed that what seemed to be changes in iPS cells' genetic makeup — presumed to be inflicted either in the course of their generation from adult cells or during their propagation and maintenance in laboratory culture dishes — instead are often accurate reflections of existing but previously undetected genetic variations among the cells comprising our bodies.

That's good news for researchers hoping to use the cells to study disease or, someday, for regenerative medicine. But it raises the question of whether and to what extent we humans are really walking mosaics whose constituent cells differ genetically from one to the next in possibly significant respects, said Alexander Urban, PhD, assistant professor of psychiatry and behavioural sciences. Urban shared senior authorship of the study with bioinformatics professor Mark Gerstein, PhD, and neurobiology professor Flora Vaccarino, MD, both of Yale.

It's only a few years ago that human iPS cells started becoming available to researchers. These cells hold great promise because they act almost exactly like embryonic stem cells, which can be nudged to differentiate into virtually any of the body's roughly 200 different cell types. But iPS cells can be derived easily from a person's skin, alleviating numerous ethical concerns arising from the necessity of obtaining embryonic stem cells from fertilized eggs.

At least in principle, iPS cells' genetic makeup closely reflects that of the individual from whom they were derived. Today, "heart cells" derived from a heart patient's skin can be produced in a laboratory dish so scientists can learn more about that particular patient's condition and to screen drugs that might treat it. Tomorrow, perhaps, such cells could be administered to that patient to restore heart health without being perceived as foreign tissue by the patient's immune system, which would otherwise reject the implanted cells.

However, Urban said, several previous studies have raised worries regarding iPS cells' genomic stability. Whether it was the reprogramming procedure researchers use to convert ordinary adult cells into iPS cells or the culturing techniques employed to keep them alive and thriving afterward, something appeared to be inducing an upswing in these cells' manifestation of copy number variations, or CNVs — the disappearance or duplication of chunks of genetic material at specific locations along the vast stretches of DNA that coil to form the chromosomes residing in all human cells.

CNVs dot everybody's genomes. They occur naturally because of DNA-copying errors made during cell replication, and accumulate in our genomes over evolutionary time. The human genome, taken as a whole, is a DNA sequence consisting of four varieties of chemical units, strung together like beads on a roughly 3-billion-bead-long necklace. CNVs range in length from under 1,000 DNA units to several million. They account for up to several percent of the entire human genome, making them a major source of genetic differences between people.

But if either iPS cells' mode of generation or their subsequent maintenance in culture were promoting an increase in CNVs, it would seriously compromise these cells' utility in research and pose a fatal flaw to their use in regenerative medicine, said Urban.

"You would never want to introduce iPS cells into a patient thinking that these cells had the same genome as the rest of the patient's cells, when in fact they had undergone substantial genetic modifications you knew nothing about, much less their effects," said Urban. (Similar concerns apply to embryonic stem cells.)

To see how serious a problem CNVs might pose for iPS cells' use, the collaborators performed tiny skin biopsies on seven volunteers and extracted cells called fibroblasts, which abound in skin and are amenable to cell culture in general and iPS cell generation in particular. From these, the team produced 20 separate iPS cell lines in culture. Using now-standard lab methods, the investigators determined, chemical unit by chemical unit, the full genomic sequence of the cells composing each new iPS cell line.

Urban and his colleagues, who had likewise assessed the fibroblasts from which the lines were derived, compared their genomic sequences with those of the newly generated iPS cells. The scientists were able to pinpoint numerous CNVs in the new cells that hadn't shown up in the fibroblasts. This raised the possibility that the rigors of reprogramming or life in a dish, or both, had led to new CNVs in the cells.

But the technique used to determine the full-length genome sequences of iPS cells and the "parental" fibroblasts from which they'd been spawned analyses not single cells but millions at a time. So a CNV residing in only a minority of cells within this mix could easily be missed, its signal swamped by the noise of the majority report.

Armed with knowledge of the precise locations along the genome where each of the "new" CNVs had popped up in their iPS cell lines, the scientists went back to the fibroblasts. This time, they used an analytical tool that, like a photocopying machine, can generate millions or billions of copies of a single section of DNA — provided that the specific DNA section is present to begin with. For each "new" CNV that had been unearthed in the iPS cells, a different version of this molecular copying machine was employed.

"Lo and behold, in many cases, this technique unearthed CNVs in the fibroblasts that were there all along but had been missed in the earlier, mass analysis," Urban said.

These CNVs had gone undetected because the fibroblasts in which they resided represented as little as a fraction of a percent of the fibroblasts in a biopsy sample. But any CNV in a fibroblast lucky enough to become the "parent" of the billions of iPS cells bearing its identical genomic "face" would now stick out like the proverbial sore thumb. Six out of the 20 different iPS cell lines sported at least one CNV unshared with the other lines, the investigators found. To the extent this reflects differences in parental fibroblasts, it means about 30 percent of the cells in human skin may harbour unique CNVs.

"This is a huge amount of cell-to-cell genetic variation," said Urban, who also participated in a recent study of human tissues conducted by the laboratory of Michael Snyder, PhD, professor and chair of genetics at Stanford. That study showed there can be distinct CNV patterns in kidney, pancreas and liver.

"Rather than monoliths, our bodies may be mosaics composed of cells whose genomes differ. What we do not know is whether or when these differences are dangerous, irrelevant or beneficial."

That may depend on the organ, said Urban.

"The more complicated something is, the more ways there are that something could go wrong with it. The brain is a particularly complicated organ. CNVs affecting cells in particular brain structures or areas could be playing a role in complex neurodevelopmental disorders such as schizophrenia and autism."

Tuesday, 6 November 2012

TIME study mirrors Late TIME trial in confirming autologous stem cells obtained from bone marrow are safe but do not further improve the recovery of heart function following a heart attack

Tuesday, 06 November 2012

Administering autologous stem cells obtained from bone marrow either 3 or 7 days following a heart attack did not improve heart function six months later, reports a new clinical trial supported by the National Institutes of Health. The results of this trial, called TIME (Transplantation in Myocardial Infarction Evaluation), were presented by Jay Traverse, MD of the Minneapolis Heart Institute Foundation Tuesday, Nov. 6, at the 2012 Scientific Sessions of the American Heart Association in Los Angeles.

The results of this trial mirror a previous, related study (LateTIME) which found that autologous bone marrow stem cell therapy given 2-3 weeks after a heart attack did not improve cardiac recovery. Both TIME and LateTIME were carried out by the Cardiovascular Cell Therapy Research Network (CCTRN), sponsored by the NIH's National Heart, Lung, and Blood Institute.

"The data presented by TIME do much to advance stem cell therapy research," said Jay Traverse, MD of the Minneapolis Heart Institute Foundation and Principal Investigator of this study.

"While this study did not provide a demonstrated cardiac benefit after six months, we still learned a great deal. Together, TIME and Late TIME have shown that stem cell therapy is safe, and they have set a baseline in terms of quantity of stem cells, type of stem cells, and severity of heart attack."

TIME enrolled 120 volunteers (avg. age 57) between July 2008 and February 2011; the participants all had moderate to severe impairment in their left ventricle and had undergone coronary stent placement as treatment for the heart attack. The participants were randomly assigned to one of four groups: day 3 stem cell, day 3 placebo (inactive cells), day 7 stem cell, or day 7 placebo. The CCTRN researchers developed a method of processing and purifying the stem cells from the bone marrow of each volunteer to ensure everyone received a uniform dose (150 million stem cells).

Heart improvement was assessed six months after stem cell therapy by measuring the percentage of blood that gets pumped out of the left ventricle during each contraction (left-ventricular ejection fraction, or LVEF). The study found no significant differences between the change in LVEF readings at the six month follow-up in either the Day 3 or Day 7 stem cell groups compared with placebo or with each other; every group showed about a 3 percent improvement in LVEF. However, the researchers found that younger patients randomized to Day 7 had greater improvement in their LVEF compared to their placebo counterparts.

"The lack of six-month improvement seen for TIME and, prior to that, LateTIME, does not mean stem cell therapy is not a viable post-heart attack strategy," said Traverse.

"Because we have this data we can start to address some parameters; for example this therapy may work better in younger people, or maybe we need to use cells from healthy volunteers (allogeneic) since their cells may provide greater therapeutic benefit. There will also be upcoming studies using novel cell types which we look forward to using in future clinical trials."

Monday, 29 October 2012

The idea of taking a mature cell and removing its identity (nuclear reprogramming) so that it can then become any kind of cell, holds great promise for repairing damaged tissue or replacing bone marrow after chemotherapy. Hot on the heels of his recent Nobel prize Dr John B. Gurdon has published today in BioMed Central's open access journal Epigenetics & Chromatin research showing that histone H3.3 deposited by the histone-interacting protein HIRA is a key step in reverting nuclei to a pluripotent type, capable of being any one of many cell types.

All of an individual's cells have the same DNA, yet these cells become programmed, as the organism matures, into different types such as heart, or lung or brain. To achieve this different genes are more or less permanently switched off in each cell lineage. As an embryo grows, after a certain number of divisions, it is no longer possible for cells which have gone down the pathway to become something else. For example heart cells cannot be converted into lung tissue, and muscle cells cannot form bone.

One way to reprogram DNA is to transfer the nucleus of a mature cell into an unfertilized egg. Proteins and other factors inside the egg alter the DNA switching some genes on and other off until it resembles the DNA of a pluripotent cell. However there seem to be some difficulties with this method in completely wiping the cell's 'memory'.

One of the mechanisms regulating the activation of genes is chromatin and in particular histones. DNA is wrapped around histones and alteration in how the DNA is wound changes which genes are available to the cell. In order to understand how nuclear reprogramming works Dr Gurdon's team transplanted a mouse nucleus into a frog oocyte (Xenopus laevis). They added fluorescently tagged histones by microinjection, so that they could see where in the cell and nucleus these histones collected.

Prof Gurdon explained:

"Using real-time microscopy it became apparent that from 10 hours onwards H3.3 (the histone involved with active genes) expressed in the oocyte became incorporated into the transplanted nucleus. When we looked in detail at the gene Oct4, which is known to be involved in making cells pluripotent, we found that H3.3 was incorporated into Oct4, and that this coincided with the onset of transcription from the gene."

Prof Gurdon's team also found that HIRA, a protein required to incorporate H3.3 into chromatin, was also required for nuclear reprogramming.

"Manipulating the H3.3 pathway may provide a way to completely wipe a cell's 'memory' and produce a truly pluripotent cell. Half a century after showing that cells can be reprogrammed this research provides a link to the work of Shinya Yamanaka (who shared the prize), and suggests that chromatin is a sticking point preventing artificially induced reprogramming being used routinely in the clinic."

Thursday, 25 October 2012

Coaxing a humble skin cell to become a jack-of-all-trades pluripotent stem cell is feat so remarkable it was honoured earlier this month with the Nobel Prize in Physiology or Medicine. Stem cell pioneer Shinya Yamanaka, MD, PhD, showed that using a virus to add just four genes to the skin cell allowed it to become pluripotent, or able to achieve many different developmental fates. But researchers and clinicians have been cautious about promoting potential therapeutic uses for these cells because the insertion of the genes could render the cells cancerous.

Now researchers at the Stanford University School of Medicine have devised an efficient and safer way to make these induced pluripotent stem cells, or iPS cells, by using just the proteins that the genes encode.

It's not the first time such an approach has been tried. Many researchers have shown that using proteins to make a cell pluripotent, although possible, is far less efficient than the virus-based method. The unprecedented success of the Stanford researchers, however, was due to an unexpected discovery: The virus used in the original method is critical for more than just gene delivery.

"It had been thought that the virus served simply as a Trojan horse to deliver the genes into the cell," said John Cooke, MD, PhD, professor of medicine and associate director of the Stanford Cardiovascular Institute.

"Now we know that the virus causes the cell to loosen its chromatin and make the DNA available for the changes necessary for it to revert to the pluripotent state."

Cooke is the senior author of the research, which will be published in the Oct. 26 issue of Cell. Postdoctoral scholars Jieun Lee, PhD, and Nazish Sayed, MD, PhD, are co-first authors of the study.

iPS cells, which don't require human embryos, offer a possible alternative for some of the ethical dilemmas associated with stem cell research. They're created from adult cells that have already assumed a specialized function in the body. Until Yamanaka's discovery, it was thought that these cells could never revert to the pluripotent stem cell from which they originated. But Yamanaka showed that these highly specialized cells are more developmentally flexible, or plastic, than previously thought. In the presence of just four genes (identified because they are highly expressed by embryonic stem cells), they can assume the characteristics of embryonic stem cells and, under the right conditions, can become nearly any cell type.

Now Cooke's research has identified an important component of how this transformation happens.

"We found that when a cell is exposed to a pathogen, it changes to adapt or defend itself against a challenge," said Cooke.

"Part of this innate immunity includes increasing access to its DNA, which is normally tightly packaged. This allows the cell to reach into its genetic toolbox and take out what it needs to survive." It also allows the pluripotency-inducing proteins to modify the DNA and transform a skin or other specialized cell into an embryonic-stem-cell-like changeling.

Because the cells activate an immune response similar to inflammation in the presence of viral genetic material, the researchers termed the process "transflammation." They believe their finding could pave the way to the use of iPS cells in humans and shed light on the biological pathways by which pluripotency occurs.

Cooke and his colleagues began by working to optimize the use of cell-permeable proteins to reprogram adult, specialized cells to become pluripotent. They knew that the proteins made it into the cell's nucleus and that, in the laboratory, they were able to bind to the correct DNA sequences. They were also able to maintain pluripotency in cells that had been reprogrammed by other means. So why were the proteins so much more inefficient than the viral-based method?

The breakthrough came when they compared the gene expression patterns of the cells exposed to the cell-permeable proteins with those of cells infected by the gene-bearing virus: They were quite different. Cooke wondered if some property of the virus could be responsible.

The researchers repeated the experiment with the cell-permeable proteins, but also included an unrelated virus. The efficiency of the pluripotency transformation increased dramatically. Further investigation revealed that the effect was due to the activation within the cell of what is called the toll-like receptor-3 pathway; triggering the pathway with a small molecule mimicking the viral genetic material had a similar effect.

"These proteins are non-integrating, and so we don't have to worry about any viral-induced damage to the host genome," said Cooke, who also noted that the use of cell-permeable proteins can confer a greater level of control of the reprogramming process and may lead to the use of iPS cells in human therapies. It also opens up new alternatives.

"Now that we understand that the cell assumes greater plasticity when challenged by a pathogen, we can theoretically use this information to further manipulate the cells to induce direct reprogramming," said Cooke.

Direct reprogramming involves inducing a specialized cell like a skin cell to become a different type of cell, like an endothelial cell, without first going through an intermediate pluripotent state. Stanford researcher Marius Wernig, MD, PhD, used direct reprogramming to successfully transform human skin cells into functional neurons.

Thursday, 27 September 2012

Small portions of male DNA, most likely left over in a mother's body by a male foetus can be detected in the maternal brain relatively frequently, according to a report published Sep. 26 in the open access journal PLOS ONE by William Chan of Fred Hutchinson Cancer Research Center and his colleagues.

This
shows a male cell in female human brain.

Credit: Citation: Chan WFN, Gurnot C,

Montine TJ, Sonnen
JA, Guthrie KA, et al.

(2012) Male Microchimerism in the
Human

Female Brain. PLoS ONE 7(9): e45592,

doi:10.1371/journal.pone.0045592.

The process, called foetal 'microchimerism (Mc)’, is common in other tissues such as blood, but this is the first evidence of male Mc in the human female brain. Microchimerism can be both beneficial and harmful to maternal health, since it is associated with processes such as tissue repair, as well as to autoimmune diseases.

Testing for the presence of a particular region of the Y-chromosome in autopsied brain tissues, the research team discovered that 63% of their samples showed potentially long-lasting Mc in multiple brain regions. They also found that women with Alzheimer's disease (AD) had less Mc than women without the disease.

According to the authors, this result warrants further investigation because previous reports have suggested that AD may be more prevalent in women with a higher number of pregnancies compared to childless women. The researchers commented that changes to the blood-brain barrier that occur during pregnancy could facilitate the process by which Mc is acquired into the human brain.

"This is the first evidence that microchimerism can cross the blood-brain barrier to establish male foetal tissue in the human female brain" says Chan.

Tuesday, 25 September 2012

Sanford-Burnham researchers identified kinase inhibitors that lower the barrier to producing stem cells in the laboratory — cells important for disease research and drug development

Tuesday, 25 September 2012

The process researchers use to generate induced pluripotent stem cells (iPSCs) — a special type of stem cell that can be made in the lab from any type of adult cell — is time consuming and inefficient. To speed things up, researchers at Sanford-Burnham Medical Research Institute (Sanford-Burnham) turned to kinase inhibitors. These chemical compounds block the activity of kinases, enzymes responsible for many aspects of cellular communication, survival, and growth. As they outline in a paper published September 25 in Nature Communications, the team found several kinase inhibitors that, when added to starter cells, help generate many more iPSCs than the standard method. This new capability will likely speed up research in many fields, better enabling scientists around the world to study human disease and develop new treatments.

Induced pluripotent stem cells
generated

using a kinase inhibitor. Credit: Sanford-

Burnham
Medical Research Institute.

"Generating iPSCs depends on the regulation of communication networks within cells," explained Tariq Rana, Ph.D., program director in Sanford-Burnham's Sanford Children's Health Research Center and senior author of the study.

"So, when you start manipulating which genes are turned on or off in cells to create pluripotent stem cells, you are probably activating a large number of kinases. Since many of these active kinases are likely inhibiting the conversion to iPSCs, it made sense to us that adding inhibitors might lower the barrier."

According to Tony Hunter, Ph.D., professor in the Molecular and Cell Biology Laboratory at the Salk Institute for Biological Studies and director of the Salk Institute Cancer Center:

"The identification of small molecules that improve the efficiency of generating iPSCs is an important step forward in being able to use these cells therapeutically. Tariq Rana's exciting new work has uncovered a class of protein kinase inhibitors that override the normal barriers to efficient iPSC formation, and these inhibitors should prove useful in generating iPSCs from new sources for experimental and ultimately therapeutic purposes."

Hunter, a kinase expert, was not involved in this study.

The promise of iPSCs

At the moment, the only treatment option available to many heart failure patients is a heart transplant. Looking for a better alternative, many researchers are coaxing stem cells into new heart muscle. In Alzheimer's disease, researchers are also interested in stem cells, using them to reproduce a person's own malfunctioning brain cells in a dish, where they can be used to test therapeutic drugs. But where do these stem cells come from? Since the advent of iPSC technology, the answer in many cases is the lab. Like their embryonic cousins, iPSCs can be used to generate just about any cell type — heart, brain, or muscle, to name a few — that can be used to test new therapies or potentially to replace diseased or damaged tissue.

It sounds simple enough: you start with any type of differentiated cell, such as skin cells, add four molecules that reprogram the cells' genomes, and then try to catch those that successfully revert to unspecialized iPSCs. But the process takes a long time and isn't very efficient — you can start with thousands of skin cells and end up with just a few iPSCs.

Inhibiting kinases to make more iPSCs

Zhonghan Li, a graduate student in Rana's laboratory, took on the task of finding kinase inhibitors that might speed up the iPSC-generating process. Scientists in the Conrad Prebys Center for Chemical Genomics, Sanford-Burnham's drug discovery facility, provided Li with a collection of more than 240 chemical compounds that inhibit kinases. Li painstakingly added them one-by-one to his cells and waited to see what happened. Several kinase inhibitors produced many more iPSCs than the untreated cells — in some cases too many iPSCs for the tiny dish housing them. The most potent inhibitors targeted three kinases in particular: AurkA, P38, and IP3K.

Working with the staff in Sanford-Burnham's genomics, bioinformatics, animal modeling, and histology core facilities — valuable resources and expertise available to all Sanford-Burnham scientists and the scientific community at large — Rana and Li further confirmed the specificity of their findings and even nailed down the mechanism behind one inhibitor's beneficial actions.

"We found that manipulating the activity of these kinases can substantially increase cellular reprogramming efficiency," Rana said.

"But what's more, we've also provided new insights into the molecular mechanism of reprogramming and revealed new functions for these kinases. We hope these findings will encourage further efforts to screen for small molecules that might prove useful in iPSC-based therapies."

Understanding how salamanders grow new limbs provides insights into the potential of human regenerative medicine

Tuesday, 25 September 2012

Based on two new studies by researchers at the Salk Institute for Biological Studies, regeneration of a new limb or organ in a human will be much more difficult than the mad scientist and super villain, Dr. Curt Connors, made it seem in the Amazing Spider-man comics and films.

As those who saw the recent "The Amazing Spiderman" movie will know, Dr. Connors injected himself with a serum made from lizard DNA to successfully regrow his missing lower right arm - that is, before the formula transformed him into a reptilian humanoid.

Salk
research shows that in the axolotl, a Mexican
salamander, jumping genes

have to be
shackled or they might move around in the
genomes of cells in the

tissue destined to
become a new limb, and disrupt the process of
regeneration.

Credit: the Salk Institute for Biological
Studies.

But by studying a real lizard-like amphibian, which can regenerate missing limbs, the Salk researchers discovered that it isn't enough to activate genes that kick starts the regenerative process. In fact, one of the first steps is to halt the activity of so-called jumping genes.

In research published August 23 in Development, Growth & Differentiation, and July 27 in Developmental Biology, the researchers show that in the Mexican axolotl, jumping genes have to be shackled or they might move around in the genomes of cells in the tissue destined to become a new limb, and disrupt the process of regeneration.

They found that two proteins, piwi-like 1 (PL1) and piwi-like 2 (PL2), perform the job of quieting down jumping genes in this immature tadpole-like form of a salamander, known as an axolotl - a creature whose name means water monster and who can regenerate everything from parts of its brain to eyes, spinal cord, and tail.

"As complex as it already seems, it might seem a hopeless task to try to regenerate a limb or body part in humans, especially since we don't know if humans even have all the genes necessary for regeneration," says Hunter.

"For this reason, it is important to understand how regeneration works at a molecular level in a vertebrate that can regenerate as a first step. What we learn may eventually lead to new methods for treating human conditions, such as wound healing and regeneration of simple tissues."

The research team, which included investigators from other universities around the country, sought to characterize the transcriptional fingerprint emerging from the early phase of axolotl regeneration. They specifically looked at the blastema, a structure that forms at a limb's stump.

There the scientists found transcriptional activation of some genes, usually found only in germ line cells, which indicated cellular reprogramming of differentiated cells into a germ line state.

In the Development, Growth & Differentiation study, the research team, led by Wei Zhu, then a postdoctoral researcher in Hunter's laboratory, focused on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon.

LINE-1 elements are jumping genes that arose early in vertebrate evolution. They are pieces of DNA that copy themselves in two stages - first from DNA to RNA by transcription, and then from RNA to DNA by reverse transcription. These DNA copies can then insert themselves into the cell's genome at new positions.

A few years ago, Fred Gage, professor in the Laboratory of Genetics at the Salk Institute, discovered that LINE-1 elements move around during neuronal development, and may program the identities of individual neurons.

"Most of these copies appear to be 'junk' DNA, because they are defective and can never jump again," says Hunter. But all mammals, including humans, still have active LINE-1 genes, and the salamander, whose genome is 10 times larger than a human's, contains many more.

Active LINE-1 retrotransposons can keep jumping, and that was true in the developing blastema where LINE-1 jumping was dramatically switched on. But in the researchers' companion study, in Developmental Biology, they found that PL1 and PL2 switch off transcription of repeat elements, such as LINE-1.

"The idea is that in the development of germ cells, you definitely don't want these things hopping around," says Hunter.

"The mobilization of these jumping genes can introduce harmful genomic rearrangements or even abort the regeneration process."

In fact, when the researchers inhibited PL1 and PL2 activity in the axolotl limb blastema, regeneration was significantly slowed down.

"The need to switch on one set of genes to stop other genes from jumping just illustrates how amazingly difficult it would be to regenerate something as complex as a limb in humans," Hunter says.

"But that doesn't mean we won't learn valuable lessons about how to treat degenerative diseases."

Salk scientists have identified a unique molecular signature in induced pluripotent stem cells (iPSCs), "reprogrammed" cells that show great promise in regenerative medicine thanks to their ability to generate a range of body tissues.

This
shows a colony of induced pluripotent

stem
cells. Blue fluorescence indicates cell

nuclei;
red and green are markers of

pluripotency.
Credit: Courtesy of the
Salk

Institute for Biological Studies.

In this week's Proceedings of the National Academy of Sciences, the Salk scientists and their collaborators at University of California, San Diego, report that there is a consistent, signature difference between embryonic and induced pluripotent stem cells. The findings could help overcome hurdles to using the induced stem cells in regenerative medicine.

"We believe that iPSCs hold a great potential for the treatment of human patients," says Juan Carlos Izpisua Belmonte, a professor in Salk's Gene Expression Laboratory and the senior author on the paper.

"Yet we must thoroughly understand the molecular mechanisms governing their safety profile in order to be confident of their function in the human body. With the discovery of these small, yet apparent, epigenetic differences, we believe that we are now one step closer to that goal."

Embryonic stem cells (ESCs) are known for their "pluripotency," the ability to differentiate into nearly any cell in the body. Because of this ability, it has long been thought that ESCs would be ideal to customize for therapeutic uses. However, when ESCs mature into specific cell types, and are then transplanted into a patient, they may elicit immune responses, potentially causing the patient to reject the cells.

In 2006, scientists discovered how to revert mature cells, which had already differentiated into particular cell types, such as skin cells or hair cells, back into a pluripotent state. These "induced pluripotent stem cells" (iPSCs), which could be developed from the patient's own cells, would theoretically carry no risk of immune rejection.

However, scientists found that iPSCs had molecular differences from embryonic stem cells. Specifically, there were epigenetic changes, chemical modifications in DNA that might alter genetic activity. At certain points in the iPSC's genome, scientists could see the presence of different patterns of methyl groups when compared to the genomes of ESCs. It seemed these changes occurred randomly.

Izpisua Belmonte and his colleagues wanted to understand more about these differences. Were they truly random, or was there a discernible pattern?

Unlike previous studies, which had primarily analysed iPSCs derived from only one mature type of cells (mainly connective tissue cells called fibroblasts), the Salk and UCSD researchers examined iPSCs derived from six different mature cell types to see if there were any commonalities. They discovered that while there were hundreds of unpredictable changes, there were some that remained consistent across the cell types: the same nine genes were associated with these common changes in all iPSCs.

"We knew there were differences between iPSCs and ESCs," says Sergio Ruiz, first author of the paper.

"We now have an identifying mark for what they are."

The therapeutic significance of these nine genes awaits further research. The importance of the current study is that it gives stem cells researchers a new and more precise understanding of iPSCs.

Friday, 14 September 2012

Biologists reveal genes key to development of pluripotency, in single cells

Friday, 14 September 2012

Several years ago, biologists discovered that regular body cells can be reprogrammed into pluripotent stem cells — cells with the ability to become any other type of cell. Such cells hold great promise for treating many human diseases.

These induced pluripotent stem cells (iPSCs) are usually created by genetically modifying cells to overexpress four genes that make them revert to an immature, embryonic state. However, the procedure works in only a small percentage of cells.

Now, new genetic markers identified by researchers at Whitehead Institute and MIT could help make that process more efficient, allowing scientists to predict which treated cells will successfully become pluripotent.

The new paper, published in the Sept. 13 online edition of Cell, also identifies new combinations of reprogramming factors that produce iPSCs, according to the researchers.

Led by Rudolf Jaenisch, a Whitehead Founding Member and an MIT professor of biology, the study is the first to examine genetic changes that occur in individual cells as they become pluripotent. Previous studies have only looked at gene-expression changes in large populations of cells — not all of which will actually reprogram — making it harder to pick out genes involved in the process.

"In previous studies, you weren't able to detect the few cells that expressed predictive pluripotency markers. The really cool part of this study is that you can detect two or three cells that express these important genes early, which has never been done before," says Dina Faddah, a graduate student in Jaenisch's lab and one of the paper's lead authors.

The other lead author is Yosef Buganim, a postdoc at Whitehead Institute.

Single-cell analysis

In 2007, scientists discovered that adult human cells could be reprogrammed by overexpressing four genes — Oct4, Sox2, c-Myc and Klf4. However, in a population of cells in which those genes are overexpressed, only about 0.1 to 1 percent will become pluripotent.

In this
image of mouse embryonic fibroblasts

undergoing
reprogramming, each coloured dot

represents
messenger RNA associated with a

specific
gene that is active in cells being

reprogrammed.
Red dots represent mRNA for

the
gene Sall4, green is Sox2, and blue is

Fbxo15.
The researchers determined that Sox2

activates
Sall4 and then activates the

downstream
gene Fbxo15, creating a gene

hierarchy
in the later phase of reprogramming.

Credit: Dina Faddah/Whitehead
Institute.

In the new study, Jaenisch's team reprogrammed mouse embryonic fibroblast cells and then measured their expression of 48 genes known or suspected to be involved in pluripotency at several points during the process. This allowed them to compare gene-expression profiles in cells that became pluripotent, those that did not, and those that were only partially reprogrammed.

Once the reprogramming, which took between 32 and 94 days, was complete, the researchers looked for genes expressed only in the cells that ended up becoming pluripotent.

The team identified four genes that were turned on very early — around six days after the reprogramming genes were delivered — in cells that ended up becoming pluripotent: Esrrb, Utf1, Lin28 and Dppa2, which control the transcription of other genes involved in pluripotency.

The researchers also found that several previously proposed markers for pluripotency were active in cells that became only partially programmed, suggesting those markers would not be useful. With their newly discovered markers, "you can eliminate all the colonies that are not completely reprogrammed," Buganim says.

To read cells' genetic profiles so precisely, the researchers screened for genes using a microfluidic system called Fluidigm, then confirmed their results with a fluorescence imaging technique that can detect single strands of messenger RNA.

Not totally random

The findings also allowed the researchers to develop a new model for how genes interact with each other to steer cells toward pluripotency. Previously, it had been thought that reprogramming was a random process — that is, once the four reprogramming genes were overexpressed, it was a matter of chance whether they would activate the correct genes to make a particular cell pluripotent.

However, the new study reveals that only the earliest phase of the process is random. Once those chance events awaken the cell's own dormant copy of the Sox2 gene, that gene launches a deterministic pathway that leads to pluripotency.

During the early, random stage, there are probably many ways that Sox2 can be activated, Buganim says.

"Different cells will activate Sox2 in different ways," he says.

"As soon as you have a specific combination that allows the activation of Sox2, you are on the way toward full reprogramming."

The new model also predicted six combinations of factors that could activate Sox2. The researchers tested these combinations in reprogrammed cells and found that they were successful, with varying rates of efficiency.

Interestingly, they found combinations that do not include any of the original reprogramming factors. The researchers are now testing their new combinations to see if they produce healthier iPSCs. The most stringent test involves injecting iPSCs into an embryo that cannot give rise to normal cells because it has four sets of chromosomes instead of two. If a healthy animal develops from those cells, it is entirely the product of the iPSCs, demonstrating that the iPSCs were equivalent to embryonic stem cells. Most iPSCs injected into embryos do not pass this test.

New relay circuits, formed across sites of complete spinal transaction, result in functional recovery in rats

Friday, 14 September 2012

In a study at the University of California, San Diego and VA San Diego Healthcare, researchers were able to regenerate "an astonishing degree" of axonal growth at the site of severe spinal cord injury in rats. Their research revealed that early stage neurons have the ability to survive and extend axons to form new, functional neuronal relays across an injury site in the adult central nervous system (CNS).

The study also proved that at least some types of adult CNS axons can overcome a normally inhibitory growth environment to grow over long distances. Importantly, stem cells across species exhibit these properties. The work will be published in the journal Cell on September 14.

The scientists embedded neural stem cells in a matrix of fibrin (a protein key to blood clotting that is already used in human neuron procedures), mixed with growth factors to form a gel. The gel was then applied to the injury site in rats with completely severed spinal cords.

"Using this method, after six weeks, the number of axons emerging from the injury site exceeded by 200-fold what had ever been seen before," said Mark Tuszynski, MD, PhD, professor in the UC San Diego Department of Neurosciences and director of the UCSD Center for Neural Repair, who headed the study.

"The axons also grew 10 times the length of axons in any previous study and, importantly, the regeneration of these axons resulted in significant functional improvement."

In addition, adult cells above the injury site regenerated into the neural stem cells, establishing a new relay circuit that could be measured electrically.

"By stimulating the spinal cord four segments above the injury and recording these electrical stimulation three segments below, we detected new relays across the transaction site," said Tuszynski.

To confirm that the mechanism underlying recovery was due to formation of new relays, when rats recovered, their spinal cords were re-transected above the implant. The rats lost motor function – confirming formation of new relays across the injury.

The grafting procedure resulted in significant functional improvement: On a 21-point walking scale, without treatment, the rats score was only 1.5; following the stem cell therapy, it rose to 7 – a score reflecting the animals' ability to move all joints of affected legs.

Results were then replicated using two human stem cell lines, one already in human trials for ALS.

"We obtained the exact results using human cells as we had in the rat cells," said Tuszynski.

The study made use of green fluorescent proteins (GFP), a technique that had never before been used to track neural stem cell growth.

"By tagging the cells with GFP, we were able to observe the stem cells grow, become neurons and grow axons, showing us the full ability of these cells to grow and make connections with the host neurons," said first author Paul Lu, PhD, assistant research scientist at UCSD's Center for Neural Repair.

"This is very exciting, because the technology didn't exist before."

According to the researchers, the study makes clear that early-stage neurons can overcome inhibitors present in the adult nervous system that normally work to maintain the elaborate central nervous system and to keep cells in the adult CNS from growing aberrantly.